According to 2017 WHO global hepatitis report, world-wide there are 257 million people carrying hepatitis B virus (HBV). HBV infection has been known to cause a series of liver disease, including liver inflammation, liver cirrhosis, and hepatocelllular carcinoma (HCC), etc., in which HBV X protein (HBx) plays an important role.
HBx when expressed in bacteria is insoluble. Dong Liu et al. developed a method for obtaining soluble HBx by using maltose binding protein tag (MBP-tag), eliminating the need for denaturation and renaturation (Biotechnol. Appl. Biochem. 2009, 54:141-147). The disadvantage of their method is its complexity. It requires amylose resin and Q-Sepharose chromatography for purification, and Factor Xa enzyme digestion for 48 hrs to remove the MBP-tag. These steps add to the complexity of the purification process, increasing cost and time and limiting its use in large-scale productions. Others have prepared HBx protein by denaturing and renaturing inclusion bodies, then metal affinity chromatography (with Ni2+ sepharose column). This method has problems of Nickel toxicity (Forgacs Z et al. (2012), J Environ Sci Health A Tox Hazard Subst Environ Eng, 47(9): 1249-60), thus limiting its use in drug manufacturing processes.
U.S. Pat. No. 9,481,714 B2 discloses a fusion protein RAP1-CD28convPEt-HBx1-154-K3, which had a similar problem with aggregate formation. A high concentration of urea was needed during the process of making the RAP1-CD28convPEt-HBx1-154-K3 for destroying hydrogen bonds within the protein to denature and solubilize the recombinant protein inside the inclusion bodies. However, a final removal of urea to refold the RAP1-CD28convPEt-HBxt1-154-K3 could easily cause formation of aggregates and thus precipitation, leading to a poor yield.
There is therefore a need to develop a novel therapeutic HBV vaccine and new method of preparing it.